Summary Our goal is to establish tools to directly label actin in any model system that can utilize genetic code expansion. Although we know a great deal about actin and the molecular components of cytoskeletal structures, we still know very little about actin dynamics that are essential to the functions of these structures. Our ability to establish mechanistic understandings of actin structures is fundamental to our knowledge of cell biology and human disease. We are limited by the availability of research tools for quantitative measurement of in vivo actin dynamics. Pinpointing a position on actin that will tolerate change is not easy due to the extensive intrafilament interfaces and the surfaces that interact with the >100 actin binding proteins. Genetic tags as small as 12 amino acids disrupt multiple cellular processes. In response to this need, we propose to take advantage of the exciting new capabilities of genetic code expansion and recently published high resolution structures of actin filaments. We will use orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs to site-specifically incorporate non-canonical amino acids (ncAAs) with reactive side chains at carefully chosen positions on actin. Using the inverse demand Diels-Alder reaction (a significantly faster variant of metal-free click chemistry) we will add fluorophores to the ncAA for in vivo imaging. We expect to be able to modify a single amino acid within actin, without disrupting function, based on the fact that actin covalently labeled with a small fluorescent probe is functional. Further, previous work shows that labeling only ~2% of actin is sufficient for visualization of most structures. Thus slight perturbations and/or low incorporation efficiency will not be a hindrance to proof-of- principle experiments. First, we will identify candidate positions for ncAA incorporation using a genetic screen. Initially, we will work in the powerful genetic model organism budding yeast, Saccaromyces cerevisiae. Because of its 87% sequence identity with skeletal actin, yeast actin has been studied for decades, providing extensive data about surface residues and powerful, yet simple, assays of actin function. Genetic code expansion is established in yeast; and, importantly, in the context of this grant, yeast work is fast. Once we have established proof-of-principle, we will shift to the fruit fly, Drosophila melanogaster. The fly is another powerful model organism that offers a broad range of genetic tools. Genetic code expansion has been demonstrated in both Drosophila-derived S2 cells and the fly. Being able to work in a relatively high throughput manner with S2 cells before moving to whole animals makes Drosophila an ideal system in which to expand. Success will result in a strategy to directly label actin in essentially every model system and tools already working in yeast and S2 cells. Success in labeling actin, will lead to major advances in our understanding of its dynamics within the cell, and provide a much needed tool to study the vast array of actin structures essential to life and health. The methodology will also provide a new approach to study closely related actin isoforms, the distinct roles of which remain poorly understood.